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Transposon Mutagenesis Paired With Deep Sequencing Of Caulobacter Crescentus Under Uranium Stress Reveals Genes Essential For Detoxification And Stress Tolerance

The ubiquitous aquatic bacteriumCaulobacter crescentusis highly resistant to uranium (U) and facilitates U biomineralization and thus holds promise as an agent of U bioremediation. To gain an understanding of howC. crescentustolerates U, we employed transposon (Tn) mutagenesis paired with deep sequencing (Tn-seq) in a global screen for genomic elements required for U resistance. Of the 3,879 annotated genes in theC. crescentusgenome, 37 were found to be specifically associated with fitness under U stress, 15 of which were subsequently tested through mutational analysis. Systematic deletion analysis revealed that mutants lacking outer membrane transporters (rsaFaandrsaFb), a stress-responsive transcription factor (cztR), or a ppGpp synthetase/hydrolase (spoT) exhibited a significantly lower survival rate under U stress. RsaFaand RsaFb, which are homologues of TolC inEscherichia coli, have previously been shown to mediate S-layer export. Transcriptional analysis revealed upregulation ofrsaFaandrsaFbby 4- and 1-fold, respectively, in the presence of U. We additionally show thatrsaFamutants accumulated higher levels of U than the wild type, with no significant increase in oxidative stress levels. Our results suggest a function for RsaFaand RsaFbin U efflux and/or maintenance of membrane integrity during U stress. In addition, we present data implicating CztR and SpoT in resistance to U stress. Together, our findings reveal novel gene targets that are key to understanding the molecular mechanisms of U resistance inC. crescentus.IMPORTANCECaulobacter crescentusis an aerobic bacterium that is highly resistant to uranium (U) and has great potential to be used in U bioremediation, but its mechanisms of U resistance are poorly understood. We conducted a Tn-seq screen to identify genes specifically required for U resistance inC. crescentus. The genes that we identified have previously remained elusive using other omics approaches and thus provide significant insight into the mechanisms of U resistance byC. crescentus. In particular, we show that outer membrane transporters RsaFaand RsaFb, previously known as part of the S-layer export machinery, may confer U resistance by U efflux and/or by maintaining membrane integrity during U stress.

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